Nirmatrelvir-resistant SARS-CoV-2 variants with high fitness in an infectious cell culture system

The oral protease inhibitor nirmatrelvir is of key importance for prevention of severe coronavirus disease 2019 (COVID-19). To facilitate resistance monitoring, we studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) escape from nirmatrelvir in cell culture. Resistant variants harbored combinations of substitutions in the SARS-CoV-2 main protease (Mpro). Reverse genetics revealed that E166V and L50F + E166V conferred high resistance in infectious culture, replicon, and Mpro systems. While L50F, E166V, and L50F + E166V decreased replication and Mpro activity, L50F and L50F + E166V variants had high fitness in the infectious system. Naturally occurring L50F compensated for fitness cost of E166V and promoted viral escape. Molecular dynamics simulations revealed that E166V and L50F + E166V weakened nirmatrelvir-Mpro binding. Polymerase inhibitor remdesivir and monoclonal antibody bebtelovimab retained activity against nirmatrelvir-resistant variants, and combination with nirmatrelvir enhanced treatment efficacy compared to individual compounds. These findings have implications for monitoring and ensuring treatments with efficacy against SARS-CoV-2 and emerging sarbecoviruses.


Mpro structure, function and inhibition
Mpro (nsp5) is one of two cysteine proteases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The function of Mpro is to cleave the SARS-CoV-2 polyproteins pp1a and pp1ab into mature nonstructural proteins at 11 cleavage sites (50). The crystal structure of Mpro with a nsp4/nsp5 peptide bound (33) shown in fig. S3a illustrates the nomenclature of the cleavage junction with P5-P1 corresponding to the five C-terminal residues of nsp4 and P1'-P2' corresponding to the two N-terminal residues of nsp5. The cleavage occurs between P1 and P1' S3b). Nirmatrelvir is a covalent inhibitor of the Mpro, which has been designed to mimic substrate residues P4-P1 (figs. S3c and S3d) (51).
Structures of Mpro with substrate peptides bound suggest that peptide bond cleavage is initiated by a nucleophilic attack on the carbonyl carbon ( fig. S3b) by the C145 thiolate, which is stabilized by the adjacent H41 (52). The attack leads to formation of a high-energy transition state with a negatively charged carbonyl oxygen (53), which is stabilized by hydrogen bonds to mainchain amide hydrogens of G143, S144 and C145 forming the oxyanion hole (fig. S13a) (33).
For nirmatrelvir, a nucleophilic attack on the nitrile warhead by the C145 thiolate leads to a reversible covalent bond to C145 and thus Mpro inhibition (32). Similarly to peptide bond breakage in natural substrates, the nucleophilic attack leads to high-energy transition states with a negatively charged nitrile nitrogen (54), which is stabilized in the structure by hydrogen bond interactions in the oxyanion hole with mainchain amide hydrogens of G143, S144 and C145 (fig. S13b).

Nirmatrelvir resistance associated substitutions
In vitro, Mpro substitutions E166V and L50F+E166V are found to confer a high level of resistance to nirmatrelvir. E166 is a key residue in Mpro connecting the substrate binding site with the dimer interface (22). In the substrate binding site, E166 stabilizes substrate binding by mainchain interactions with P3 and side chain interactions to the amide nitrogen in the P1 glutamine ( fig.   S13a). These interactions are maintained when nirmatrelvir binds (fig. S13b). E166 furthermore interacts with the N-terminal S1 in the other Mpro monomer, and is involved in substrate-induced Mpro dimerization (22). As Mpro monomers are not catalytically active, dimerization is essential for Mpro function (55).
Additionally, the S1-E166 interaction is essential for maintaining the correct shape of subsite S1 (50). This is supported by several studies of highly similar SARS-CoV Mpro variants. In one study, it was found that deletion of the N-terminal residues 1-3 in the SARS-CoV Mpro reduced the catalytic efficiency to 76% compared to the original Mpro and led to a slight reduction in dimerization (56). In comparison, deletion of N-terminal residues 1-4 led to a dramatic shift of the Mpro monomer-dimer equilibrium resulting in the monomer being the major species and led to a reduction of the catalytic efficiency to 1% (56). Another study found that the mutation S1A did not affect the monomer-dimer equilibrium and resulted in 46% catalytic efficiency compared to original Mpro (57). A third study found that E166A slightly reduced Mpro dimerization and reduced catalytic efficiency to 31% compared to the original Mpro. In combination with another mutation in the dimer interface, R298A, E166A shifted the Mpro monomer-dimer equilibrium towards monomer as the major species (22).
The results in these studies highlight the important role of the S1-E166-substrate interaction for maintaining the enzymatic activity of Mpro. The reduced enzymatic activity upon breakage of the S1-E166-substrate interaction is suggested to result from a disruption of the correct catalytically competent conformation of the substrate binding site including the oxyanion hole (18) and a reduction in dimerization (22).

Trajectory analysis for simulations
The root-mean-square deviation (RMSD) evolutions of the nirmatrelvir and nsp4/nsp5 substrate peptide extracted from the simulations are shown in fig. S4 and fig. S5, respectively. The RMSD values fluctuate around average values ranging from 1.8-2.4 Å indicating that the Mpro dimer is stable throughout the simulations and that the simulations have converged within 50 ns. We used the last 50 ns of the simulations for further analyses.

Nirmatrelvir-Mpro interactions
To evaluate how the interactions between nirmatrelvir and the SARS-CoV-2 Mpro changed for the

Interaction energy analysis
The interaction energies extracted from the Mpro-nirmatrelvir and Mpro-substrate peptide MD simulations are presented in table S3, and interaction energy differences are presented in Fig. 7A.
The relatively large errors for the substrate peptide simulations reflect the flexibility of the nsp4/nsp5 peptide.
For Mpro with E166V or L50F+E166V, we observe less favourable interaction energies for nirmatrelvir relative to original Mpro resulting mainly from reduction in the coulombic contribution to the interaction energies. This may be explained by the loss of S1-E166-nirmatrelvir interaction

Inhibition probabilities
For nirmatrelvir to successfully inhibit the SARS-CoV-2 Mpro, nirmatrelvir must bind to the Mpro in a conformation that is compatible with catalysis and thus leads to covalent attachment of nirmatrelvir to Mpro C145. We define a catalytically competent state by two criteria, 1) the sulfur atom in C145 and cyano carbon atom in nirmatrelvir should be in close enough proximity for the nucleophilic attack (54), and 2) the backbone amide of G143 in the oxyanion hole and cyano nitrogen atom in nirmatrelvir should be in close enough proximity for stabilizing the nirmatrelvir transition state (18). The distances are shown in the Mpro structure in fig For the L50F variant, there is a slight decrease in the probability of nirmatrelvir-Mpro being in a catalytically competent conformation relative to original Mpro (24% to 20%). The probability for E166V to be in a catalytically competent state is reduced to 50% compared to the original Mpro (from 24% to 12%), while a third population with larger G143 amide nitrogen-nirmatrelvir cyano nitrogen distances is present for the L50F+E166V variant thus reducing the probability of a catalytically competent state to 63% compared to the original Mpro (from 24% to 15%).

Impact of mutations on dimerization and integrity of subsite S1
L50F, E166V, and L50F+E166V led to improved or similar Mpro-substrate binding compared to the original Mpro (Fig. 7A). Thus, weakening of substrate binding cannot explain the reduced Mpro activity and replication observed in our experiments (Figs. 4 to 6). Instead, analysis of the Mprosubstrate MD simulations suggests that perturbation of subsite S1 and destabilization of the Mpro dimer may explain the reduced catalytic activity.
The Mpro exists in two conformations corresponding to an active and an inactive state. The active state of subsite S1 is stabilized by hydrophobic interactions between F140 and H163 (21). We have monitored the distribution of distances between F140 and H163, and their orientation to each other.
The latter was defined as the dihedral angle between the phenyl ring of F140 and the imidazole ring of H163. fig. S10 displays the 2D scatter plots of dihedral angle vs. F140-H163 distance for the different variants (original Mpro, E166V, L50F+E166V and L50F). For original Mpro, the most populated state is observed when the distance and dihedral angle between the two side chains is around 4 Å and 0 degrees, respectively, indicating that the side chains are packed face-to-face. This agrees well with other MD simulations on original Mpro in the active conformation (21). The distributions for the F140-H163 interaction in E166V and L50F+E166V are broader in comparison to the distributions recorded for original Mpro indicating that the S1 binding site is more flexible in these variants. This results in the impairment of the hydrophobic stacking between F140 and H163 and consequently inducing structural perturbation leading to the distortion of the active conformation. Even though the substrate can still bind to E166V and L50F+E166V, the Mprosubstrate complex is perturbed leading to an increase in the population of an inactive state of Mpro.
For L50F, the distance and orientation between F140 and H163 side chains are not affected by the mutation, resulting in 2D scatter plot resembling the one observed for original Mpro. This observation cannot explain the low activity measured for this variant. We therefore focused on the stability of the Mpro dimer since dimerization is a requirement for Mpro to be catalytically active.
We have evaluated the effect of the mutations on dimerization by monitoring the dimer interactions, S1-F140 and R4-E290, which are important for dimer stability (20). The results are presented in fig.   S9 displaying the 2D scatter plots of the distance R4-E290 vs. S1-F140 for the different variants.
For original Mpro, the mean S1-F140 distance is 2.8 Å  0.1 Å, whereas the mean R4-E290 distance is 3.4 Å  1.3 Å. Contrarily, for the variants, relatively large fluctuations in both distances are observed that contribute to the destabilization of the dimer and hence impairment of activity.
g Specification of conditions under which viral genomes subjected to NGS were sampled. Fold EC50 applied and passage (P) and/or day (D) postinfection at sampling time.